U.S. patent number 6,617,107 [Application Number 09/601,599] was granted by the patent office on 2003-09-09 for specific oligonucleotide primers for detection of bovine male chromosome presence by polymerase chain reaction and method.
This patent grant is currently assigned to XY, Inc.. Invention is credited to Alan D. Dean.
United States Patent |
6,617,107 |
Dean |
September 9, 2003 |
Specific oligonucleotide primers for detection of bovine male
chromosome presence by polymerase chain reaction and method
Abstract
This invention is a specific set of oligonucleotide Polymerase
chain reaction (PCR) primers [5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ ID
No. 1) and 5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No. 2)] for specific
polymerase chain reaction (PCR) amplification of a region of the
bovine male-specific chromosome sequence.
Inventors: |
Dean; Alan D. (Fort Collins,
CO) |
Assignee: |
XY, Inc. (Fort Collins,
CO)
|
Family
ID: |
22116263 |
Appl.
No.: |
09/601,599 |
Filed: |
August 3, 2000 |
PCT
Filed: |
February 03, 1999 |
PCT No.: |
PCT/US99/02387 |
PCT
Pub. No.: |
WO99/38883 |
PCT
Pub. Date: |
August 05, 1999 |
Current U.S.
Class: |
435/6.15;
435/91.1; 435/91.2; 536/22.1; 536/24.3; 536/24.31 |
Current CPC
Class: |
C12Q
1/6879 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12B 001/68 (); C12P 019/34 ();
C07H 021/00 (); C07H 021/04 () |
Field of
Search: |
;435/91.1,91.2,6
;536/22.1,24.3,24.31 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
The nucleic acid sequence search report, Accesion No. U75895.*
.
Vogel, Tanja; Deschend, Frank; Manz, Eberhard; Jung, Christian;
Jakubiczka, Sybille; Fehr, Susanne; Schmidtke, Jorg; Schnieders,
Frank;"Organization and expression of bovine TSPY ", Mammalian
Genome 8, 491-496 (1997)..
|
Primary Examiner: Whisenant; Ethan
Assistant Examiner: Tung; Joyce
Attorney, Agent or Firm: Santangelo Law Offices, P.C. Miles;
Craig R.
Parent Case Text
This application is the National Stage of International Application
No. PCT/US99/02387, filed Feb. 3, 1999 which claims the benefit of
U.S. Provisional Application No. 60/073,863, filed Feb. 3, 1998,
each hereby incorporated by reference.
Claims
What is claimed is:
1. A polymerase chain reaction primer the nucleotide sequence of
which consists of 5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ ID NO:1).
2. A polymerase chain reaction primer the nucleotide sequence of
which consists of 5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID NO:2).
3. A primer pair used for amplifying DNA of the bovine TSPY gene by
polymerase chain reaction, wherein the primer pair comprises a
first primer comprising the nucleotide sequence of
5'-TGATCCGGCATATAGCTGAGA-3' (SEQ ID No. 1), and a second primer the
nucleotide sequence of 5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No.
2).
4. The primer pair used for amplifying DNA of the bovine TSPY gene
by polymerase chain reaction according to claim 3, wherein said
bovine TSPY gene comprises Bos taurus TSPY gene.
5. A kit useful to detect the presence of a portion of DNA sequence
from Y chromosome, comprising: a a first container containing a
first oligonucleotide primer comprising the nucleotide sequence
5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ ID No. 1), wherein said first
oligonucleotide primer binds to a specific Y chromosome
polynucleotide sequence; and b at least a second container
containing a reagent useful in performance of a polyrnerase chain
reaction amplification.
6. A kit useful to detect the presence of a portion of DNA sequence
from Y chromosome, comprising: a a first container containing a
second oligonucleotide primer comprising the nucleotide sequence
5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No. 2), wherein said second
oligonucleotide primer binds to a specific Y chromosome
polynucleotide sequence; and b at least a second container
containing a reagent useful in performance of a polymerase chain
reaction amplification.
7. A kit useful to detect the presence of a portion of DNA sequence
from Y chromosome according to claims 5 or 6, wherein said first
container contains both said first oligonucleotide primer (SEQ ID
No. 1) and said second oligonucleotide primer (SEQ ID No. 2), and
wherein said first oligonucleotide primer (SEQ ID No. 1) and said
second oligonucleotide primer (SEQ ID No. 2) each bind to a
specific Y chromosome polynucleotide sequence.
8. A kit useful to detect the presence of a portion of DNA sequence
from Y chromosome according to claim 5, wherein said at least
second container contains a DNA polymerase enzyme.
9. A kit useful to detect the presence of a portion of DNA sequence
from Y chromosome according to claim 5, further comprising a third
container containing a reagent to detect said specific Y chromosome
polynucleotide sequence after amplification.
10. A method of detecting a specific Y chromosome sequence by
polymerase chain reaction, comprising the steps of: a amplifying a
specific Y chromosome polynucleotide sequence with a primer pair,
wherein said primer pair comprises a first primer comprising the
nucleotide sequence of 5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ ID No. 1)
and a second primer comprising the nucleotide sequence of
5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No. 2); and b detecting the
polynucleotide product of said step of amplifying said specific Y
chromosome polynucleotide.
11. A method of identifying the gender of an animal, comprising the
steps of: a obtaining a meat sample from said animal; b amplifying
a target nucleic acid sequence in said meat with a primer
comprising the nucleotide sequence of 5'-GTGATCCGGCATATAGCTGAGA-3'
(SEQ ID No. 1); c isolating amplified products from said genetic
material; d determining the existence of Y chromosome
polynucleotide sequence in said meat.
12. The method of identifying the gender of an animal according to
claim 11, further comprising the step of amplifying said a second
target nucleic acid sequence in said meat with a primer comprising
the nucleotide sequence of 5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No.
2).
13. The method of identifying the gender of an animal according to
claims 11 or 12, further comprising the step of amplifying the said
first target nucleotide sequence and said second nucleotide
sequence with a primer pair, wherein said primer pair comprises a
first primer comprising the sequence of
5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ ID No. 1) and a second primer
comprising the sequence of 5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No.
2).
14. A method of amplifying a specific Y chromosome polynucleotide
sequence by polymerase chain reaction, comprising the steps of: a
establishing a specific Y chromosome polynucleotide sequence in a
solution; b providing a primer pair, wherein said primer pair
comprises a first primer comprising the sequence of
5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ ID No. 1) and a second primer
comprising the sequence of 5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No.
2) in said solution; c denaturing said specific Y chromosome
sequence; d annealing each of said primer pair to a specific region
on said specific Y chromosome sequence; and e extending each of
said primer pair on said specific Y chromosome sequence using said
specific Y chromosome sequence as a template to amplify a portion
of said Y chromosome sequence.
15. The method of amplifying a specific Y chromosome sequence by
polymerase chain reaction according to claim 7, further comprising
repeating the steps of denaturing said specific Y chromosome
sequence, annealing each of said primer pair to a specific region
on said specific Y chromosome sequence, and extending each of said
primer pair on said specific Y chromosome sequence using said
specific Y chromosome sequence as a template to amplify a portion
of said Y chromosome sequence.
16. A method of identifying the gender of an animal by polymerase
chain reaction, comprising the steps of: a establishing a DNA
material from a sample of meat of said animal in a solution; b
providing a primer pair, wherein said primer pair comprises a first
primer comprising the sequence of 5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ
ID No. 1) and a second primer comprising the sequence of
5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No. 2) in said solution; c
denaturing said DNA material to produce two single stranded DNA
sequences; d annealing each of said primer pair to a specific
region on each of said single stranded DNA sequences which
sequences are complementary to each of said primer sequences; e
extending each of said primer pair on each of said single stranded
DNA sequences using each of said single stranded DNA sequences as
templates to amplify a specific region of each of said single
stranded DNA sequences; f repeating said steps of denaturing said
DNA material to produce two single stranded DNA sequences;
annealing each of said primer pair to a specific region on each of
said single stranded DNA sequences which sequences are
complementary to each of said primer sequences; extending each of
said primer pair on each of said single stranded DNA sequences
using each of said single stranded DNA sequences as templates to
amplify a specific region of each of said single stranded DNA
sequences; and g detecting the presence of said specific regions
from said DNA material wherein said specific regions have been
amplified following said steps of c, d and e and wherein the
presence of said specific regions from said DNA material is
indicative of male genomic material in said DNA material.
Description
I. TECHNICAL FIELD
This invention relates to a specific set of primers for uniquely
determining the presence of bovine Y chromosome. Specifically, it
includes unique primers for the amplification by a polymerase chain
reaction (PCR) methodology of a specific bovine Y chromosome
sequence. This invention also relates to a method for detection of
the presence of a bovine male chromosome sequence using specific
oligonucleotide primers for amplification of the male chromosome
sequence by polymerase chain reaction.
II. BACKGROUND
The methodology of polymerase chain reaction (PCR) was invented by
Kary Mullis in the mid-1980s and has revolutionized molecular
genetics by making possible a whole new approach to the study and
analysis of genes. The methodology has become well known since it
was invented. PCR allows amplification of a preselected region of
DNA and can serve as a highly specific and sensitive detection
method (K. B. Mullis and F. A. Faloona, Methods Enzymol.
155:335-350, 1987). Some specific PCR methods have been patented in
such patents as U.S. Pat. Nos. 4,683,195 and No. 4,683,202 to
Mullis, et al. which are hereby incorporated by reference.
The PCR has also been used for the identification of organisms in
complex substrates and to the detection of infectious agents (D. M.
Olive, J. Clin. Microbiol. 27: 261-265; B. I. Eisenstein, New Engl.
J. Med. 322: 178-183, 1990). Some specific oligonucleotide primers
have been developed for detection of pathogenic bacteria by PCR
(U.S. Pat. No. 5,494,795). Because of its efficiency and
specificity, PCR techniques have been applied extensively to every
field in which the molecular techniques are employed and to the
detection of presence of a specific gene or a portion of the gene
of interest.
Efforts of use of PCR have also been made in the meat and dairy
industry. It is practically important to recognize the gender in
some applications by detecting the presence of the unique Y
chromosome in male cells or tissues. The identification of the
presence of any portion of a male chromosome can be important for a
number of reasons. One of the reasons is the identification of
tissues or meat by gender after the tissues have been removed from
the animal and disbursed through marketing channels. By identifying
for instance the gender (bull or cow in this case), a determination
of proper purchases, representations, and quality of the tissues
maybe made. Therefore, efforts of designing appropriate primers
have been undertaken and a pair of primers specifically identifying
a portion of bovine Y chromsome has been successfully designed by
the present inventor. Several aspects of the general bovine
sequence had been previously identified prior to the present
invention. However, among the aspects unique to this invention are
the particular and specific primers complementary to the particular
sequence of the bovine male chromosome. Despite attempts by others
in the art to test or probe for the Y chromosomes, apparently it
escaped those in the field to create such primers uniquely designed
to amplify the specific region of the sequence on the Y chromosome
to indicate the gender. While other pruners may exist in the
marketplace, these primers are unique. By simply using these
primers on the tissues, a ready identification of the gender may be
made. The prior references do not teach effective or suitable
primers to the extent now shown.
III. DISCLOSURE OF THE INVENTION
Accordingly, an object of this invention is a set of
oligonucleotide primers [sequence 5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ
ID No. 1)] and [sequence 5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No.
2)] for PCR amplification of a portion of bovine Y chromosome DNA
sequence.
An additional object of this invention is the demonstration of the
specificity of the above oligonucleotide primers for use in
detecting the presence of a portion of bovine Y chromosome DNA
sequence. This demonstration is specific only to a region of the Y
chromosome sequences in bulls but not specific to other chromosome
DNA sequences in bulls and chromosome sequences in cows.
These and additional objects of the invention are accomplished by
application of standard PCR methodology employing the
oligonucleotide primers [5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ ID No.
1) and 5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No. 2)] to amplify a
portion of the bovine Y chromosome DNA sequence. The bovine species
used in this invention in particular is Bos taurus but these
primers might be used in other bovine species and, to-some extent,
even in species other than bovines.
IV. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the electrophoretic results of the amplification
products obtained using Y-specific primers.
FIG. 2 shows the electrophoretic results of the amplification
products obtained using primers specific for the bovine prion gene
as a control.
V. SEQUENCE LISTING FREE TEXT 1. The first nucleotide sequence is a
forward primer 5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ ID) No. 1). 2. The
second nucleotide sequence is a reverse primer
5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No. 2).
VI. BEST MODE FOR CARRYING OUT THE INVENTION
As can be easily understood, the basic concepts of the present
invention may be embodied in a variety of ways. It involves
techniques as well as devices to accomplish the appropriate
invention. In this application, the techniques are disclosed as
part of the results shown to be achieved by the various sequences
described and as steps which are inherent to utilization. They are
simply the natural result of utilizing the sequences as intended
and described. In addition, while the sequences are disclosed, it
would be understood that these not only accomplish certain methods
but also can be varied in a number of ways. Importantly, as to all
of the foregoing, all of these facets should be understood to be
encompassed by this disclosure.
In the preferred embodiment, the present invention uses PCR
technique to amplify a portion of a particular bovine sequence.
This sequence may be a portion the Bos taurus testis-specific
protein, Y-encoded (TSPY) gene. However, other sequences than
bovine sequences might be identified using these primers, and so
the invention may not be restricted to just bovine sequences. This
invention has uniquely identified two primers that are
complementary to this bovine sequence found only in the Y
chromosome, the Bos taurus TSPY gene. While other sequences have
been used to identify other aspects of the Y chromosome, apparently
these primers are uniquely designed to identify this portion of the
Y chromosome.
The preferred embodiment of this invention is a specific set of
oligonucleotide primers ([SEQ. ID No. 1] and [SEQ. ID No. 2]) for
PCR amplification of a specific region of the bovine Y chromosome
DNA sequence. Any oligonucleotide probe may be used that is
internal to the sequence amplified by the PCR primers ([SEQ. ID No.
1] and [SEQ. ID No. 2]). The label can be any one of the art
recognized labels commonly used in DNA blotting.
Two oligonucleotides, [5'-GTGATCCGGCATATAGCTGAGA-3' (SEQ ID No. 1)
and 5'-TGGTCGCTGATCAGGATGGAA-3' (SEQ ID No. 2)], have been designed
based on the DNA sequence data of the bovine TSPY gene from
GenBank. These two oligonucleotides function as specific primers
for PCR amplification of the portion of the bovine TSPY gene. The
PCR was performed following the protocol as disclosed herein in
examples below. The results are described in detail below by the
inventor.
Having described the invention in brief, the following examples are
given to illustrate specific applications of the invention. These
specific examples are not intended to limit the scope of the
invention described in this application.
VII. EXAMPLES
The following were the amplification protocol, including the primer
sequences, the reagents and the results.
1. PCR Amplification
The reagents used in this invention were listed as follows:
Molecular grade water (Sigma W-3500); dNTPs (10 mM mix of each
deoxyribonucleoside-triphosphates); Taq DNA Polymerase (5U/.mu.l:
Sigma D-1806. 10.times.reaction buffer is included);
10.times.reaction buffer (100 mM Tris-HCl, pH 8.3, 500 nM KCl, 15
mM MgCl.sub.2); Forward primer (100 ng/.mu.l) 5'-GTG ATC CGG CAT
ATA GCT GAG A-3'; Reverse primer (100 ng/.mu.l) 5'-TGG TCG CTG ATC
AGG ATG GAA-3'; and Bovine Test DNA (10-50 ng/reaction). Master mix
for 50 .mu.l reaction was prepared following the following
recipe:
Reagents Volume (.mu.l) Water 38.75 10X Buffer 5 dNTPS 1 Forward
primer 2 Reverse primer 2 Taq polymerase 0.25 Bovine Test DNA 1
The amplification reaction parameters for the amplification cycles
were denaturation for 1 min at 94 degree(s) C., annealing of
primers for 1 min at 56 degree(s) C., and primer extension for 3
seconds at 72 degree(s) C. The thermocycler parameter settings were
as follows:
Step Temperature Time 1 94.degree. C. 30 seconds 2 94.degree. C. 30
seconds 3 56.degree. C. 30 seconds 4 72.degree. C. 30 seconds
(Repeat steps 2-4 for 30 cycles) 5 72.degree. C. 5 minutes 6
4.degree. C. Soak
2. Demonstration of Specificity
Both the forward (SEQ ID NO.1) and reverse primers (SEQ ID NO. 2)
were evaluated for their ability to amplify a portion of Y
chromosome sequence. With a bull or steer DNA a 488 base pair
product was amplified. The primers generated the
appropriately-sized fragments from DNA preparations from blood
samples of bulls when compared to the known standard DNA from
bulls. The fact that the primers did not generate a detectable PCR
product with DNA from cows suggests that the primers are
Y-chromosome sequence specific.
The above example, incorporated as if fully set forth here in text,
shows the procedure and results such that one with ordinary skill
in the art could duplicate the efforts to produce the specific
primers. As shown, the forward primer (SEQ NO. 1) is 5'-GTG ATC CGG
CAT ATA GCT GAG A-3' and the reverse primer (SEQ NO. 2) is 5'-TGG
TCG CTG ATC AGG ATG GAA-3'. Naturally, these primers may be varied
in length or other alterations as long as the functional aspects
were retained to hybridize with and to amplify the TSPY gene to
indicate gender. This could include from 8-20 nucleotides of the
sequences. These primers amplify using the PCR method approximately
488 base pair product of the Y chromosome. Naturally, as those with
ordinary skill in the art would understand, the above primer
sequences could vary to some degree such that the functional
derivative would still be specific for a portion of the Y
chromosome. Additionally, while specific reagents and buffers are
described and while proportions are disclosed, a variety of buffers
and reagents and varying proportions could be used in conjunction
with the specific primers of SEQ ID NO.1 and SEQ ID NO.2. Thus, for
example any buffering means or any buffering proportions which
produce similar or acceptable results in conjunction with SEQ ID
NO.1 and SEQ ID NO.2 to identify a specific region of the Y
chromosome should be considered included in this invention.
Similarly, any reagent means or any reagent proportions which
produce similar or acceptable results in conjunction with SEQ ID
NO.1 and SEQ ID NO.2 to identify a specific region of the Y
chromosome should be considered included in this invention. Other
parameters, such as the parameters of the thermocyler or other
similar devices, could be varied to accomplish the goals and
purposes of the invention.
The present invention is also directed to a kit or reagent system
useful for practicing the methods described herein. Such a kit will
contain a reagent combination comprising the essential elements
required to conduct an assay according to the methods disclosed
herein. The reagent system is presented in a commercially packaged
form, as a composition or admixture where the compatibility of the
reagents will allow, in a test device configuration, or more
typically as a test kit, i.e., a packaged combination of one or
more containers, devices, or the like holding the necessary
reagents, and usually including written instructions for the
performance of assays. The kit of the present invention may include
any configurations and compositions for performing the various
assay formats described herein.
In all cases, the reagent system will comprise a pair of
oligonucleotide primers that flank the DNA sequence of interest
which is to be amplified and detected, as described herein.
Preferably, those primers are (1) SEQ ID NO:1 and (2) SEQ ID NO:2
and they may be detectably labeled. Preferably, the kit will also
contain additional reagents useful in carrying out a PCR
amplification. A kit according to the present invention can
additionally include ancillary chemicals such as the buffers and
components of the solution in which binding of the oligonucleotide
to the target DNA takes place.
Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is
therefore to be understood that, within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described.
The discussion included in this application is intended to serve as
a basic description. The reader should be aware that the specific
discussion may not explicitly describe all embodiments possible;
many alternatives are implicit. The market place and manufacturing
concerns may dictate the appropriate embodiments for the present
invention. Particularly with respect to the discussion, it should
be understood that a number of changes may be made without
departing from the essence of the present invention. In this
regard, it is intended that such changes--to the extent that they
substantially achieve the same results in substantially the same
way--will still fall within the scope of the present invention. It
also may not fully explain the generic nature of the invention and
may not explicitly show how each feature or element can actually be
representative of a broader function or of a great variety of
alternative or equivalent elements. Again, these are implicitly
included in this disclosure. Where the invention is described in
sequence-oriented terminology, each element of the sequence
implicitly performs a function. Sequence discussions or claims may
not only be included for the sequence described, but also method or
process claims may be included to address the functions the
invention and each element performs. Although the methods related
to the system are being included in various detail, only an initial
discussion directed toward the sequences have been included.
Naturally, that discussion could have some application to the
various other methods and aspects discussed throughout the
disclosure. Neither the description nor the terminology is intended
to limit the scope of the claims which will be included in a full
patent application.
It should be understood that a variety of changes may be made
without departing from the essence of the invention. Such changes
are also implicitly included in the description. They still fall
within the scope of this invention. A broad disclosure encompassing
both the explicit embodiment(s) shown, the great variety of
implicit alternative embodiments, and the broad methods or
processes and the like are encompassed by this disclosure.
Each of the various elements of the invention and claims may also
be achieved in a variety of manners. This disclosure should be
understood to encompass each such variation, be it a functional
derivative, such as under high stringency, a method or process
embodiment of the sequences, or even merely a variation of any
element of these. Particularly, it should be understood that as the
disclosure relates to elements of the invention, the words for each
element may be expressed by equivalent terms or method terms--even
if only the function or result is the same. Such equivalent,
broader, or even more generic terms should be considered to be
encompassed in the description of each element or action. Such
terms can be substituted where desired to make explicit the
implicitly broad coverage to which this invention is entitled. As
but one example, it should be understood that all action may be
expressed as a means for taking that action or as an element which
causes that action. Similarly, each physical element or
compositions disclosed should be understood to encompass a
disclosure of the action which that physical element or composition
facilitates. Regarding this last aspect, the disclosure of a
"buffer" should be understood to encompass disclosure of the act of
"buffering" whether explicitly discussed or not--and, conversely,
were there only disclosure of the act of "buffering", such a
disclosure should be understood to encompass disclosure of a
"buffer." Such changes and alternative terms are to be understood
to be explicitly included in the description.
In addition, it should be understood that, in the claims and in the
application, the term "comprising" is meant to have an inclusive
meaning rather than an exclusive one. It should be interpreted in
its most expansive form so as to afford the applicant the broadest
coverage legally permissible. Therefore, in countries, such as
Australia, this term is not intended to have an exclusive, or more
limited, meaning.
Any references mentioned in the application for this patent are
hereby incorporated by reference, however, to the extent statements
might be considered inconsistent with the patenting of this
invention such statements are expressly not to be considered as
made by the applicant.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 2 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 1
gtgatccggc atatagctga ga 22 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 2 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 2
tggtcgctga tcaggatgga a 21
* * * * *